The proposed studies attack several problems related to digitalis glycosides. The first objective is the elucidation of mechanism(s) of action of the cardiac glycosides by biochemical, immunochemical, and physiological studies of the putative digitalis receptor (Na ion plus K ion)-ATPase. Solubilization and further purification of myocardial (Na ion plus K ion)-ATPase using the detergents Lubrol-WX and sodium deoxycholate will be pursued with the objective of obtaining a homogeneous preparation of high specific activity, which will facilitate studies of enzymatic mechanisms and cardiac glycoside interactions. Antibodies against (Na ion plus K ion)-ATPase have been obtained and will be used in further studies of enzyme and monovalent cation transport mechanisms; use of these antibodies as tools in the isolation and purification of solubilized (Na ion plus K ion)-ATPase and sarcolemmal membrane fragments will also be explored. Further studies will be aimed at characterization of cellular turnover (Na ion plus K ion)-ATPase. Adaptive changes in this enzyme in response to various influences will also be studied. The second broad area of effort deals with cardiac glycoside-specific antibodies. Purification of digoxin-specific antibodies and their Fab fragments has been achieved by an affinity chromatography approach. Reversal of digitoxin intoxication by digoxin-specific antibodies in canine models will be studied and analogous methods for isolation and purification of digitoxin-specific antibodies devised if necessary. Antigenicity studies in rabbit and primate (baboon) models will be completed, and clinical testing begun in selected cases of life-threatening digitalis toxicity unresponsive to conventional measures. At the fundamental level, kinetic studies of cardiac glycoside-antibody interactions will be correlated with equilibrium binding and hapten displacement experiments to define the determinants of antibody-hapten interaction specificity. BIBLIOGRAPHIC REFERENCES: Beller, G.A., Hood, W.B., Jr., and Smith, T.W.: Effects of ischemia and re-perfusion on myocardial uptake of tritiated digoxin. In Roy, P-E., Dhalla, N.S. (eds.): Recent Advances in Studies on Cardiac Structure and Metabolism. Volume 9: The Sarcolemma. Baltimore, University Park Press, 1976, pp. 475-485.